WO1996019498A2

WO1996019498A2 - T cell stimulating protein of pestivirus

Info

Publication number: WO1996019498A2
Application number: PCT/EP1995/005066
Authority: WO
Inventors: Heinz-Jurgen Thiel; Knut Elbers; Thomas Pauly
Original assignee: Akzo Nobel N.V.
Priority date: 1994-12-20
Filing date: 1995-12-20
Publication date: 1996-06-27
Also published as: US5965134A; ATE206458T1; PT772632E; HU222367B1; DE69523055T2; DK0772632T3; JPH09509682A; EP0772632A2; ES2164169T3; HUT75376A; WO1996019498A3; EP0772632B1; HU9601981D0; DE69523055D1

Abstract

This application is concerned with immunogenic polypeptides of pestiviruses, specifically Classical Swine Fever Virus (CSFV), specifically the nonstructural protein p10, more specifically T cell epitopes from this protein and nucleic acid molecules encoding these polypeptides, and vaccines and diagnostics with these polypeptides or nucleic acid molecules.

Description

T CELL STIMULATING PROTEIN OF PESTIVIRUS

This application is concerned with polypeptides comprising T lymphocyte stimulatory proteins of pestiviruses, especially Classical Swine Fever Virus (CSFV), specifically the nonstructural protein p10, more specifically T cell epitopes from this protein, and nucleic acids coding for these polypeptides, recombinant vectors with these nucleic acids, host cells transformed with these vectors, vaccines and diagnostics with this polypeptides and processes for preparing them.

The genus pestivirus belongs to the family of Flaviviridae and consists of Classical Swine Fever Virus (or hog cholera virus), which is the causative agent of classical swinefever, bovine viral diarrhoea virus (BVDV) infectious to cattle and border disease virus (BDV) infectious to sheep.

Pestiviruses are small enveloped RNA viruses having a diameter of 40-60 run. The virions consist of a nuclear capsid enveloped by a lipid layer embedded with glycoproteins. The genome of pestiviruses consists of a single stranded RNA of approximately 12.5 kB. It contains a single open reading frame (ORF) which is flanked by parts that are not being translated. The 3' end is not polyaαenyiated.

Viral proteins are formed by co- and posttranslational processing of a hypothetic polyprotein, while the structural proteins are coded in the 5' end of the genome. In contrast to other flaviviridae the pestiviruses have a sequence coding for a non structural p23 protein at the 5' end. This N-terminal protease (Npro) is spliced from the next protein by an autoproteolytical process. In the 3' direction the sequence coding for a p14 nuclear capsid and a signal sequence, which is responsible for the translocation of the subsequent sequences for glycoprotein E0, E1 and E2 in the lumen of the endoplasmatic reticulum (ER), are following. The splicing of the single glycoproteins probably is caused by cellular signalases in the ER. The glycoproteins can form complex structures in infected cells by dimensioning through S-S-bridges. The function of these complex structures is not yet known.

Next to the sequences for the structural proteins the sequences for the non structural proteins p125, p10, p30 and p133 are* found in the polyprotein. Analogous to posttranslational processes in cytopathogenic Bovine Viral Diarrhea Virus (BVDV, another pestivirus) an 80p protein can be detected after processing the p125 protein (Desport, M. and Brownlie J., Arch. Virol. Suppl. 3, 261-265, 1991). The p133 non structural protein, which is processed into p58 and p75 proteins, contains sequence motifs which resemble RNA polymerase sequences. The amino acid sequences of the non-structural CSFV proteins are considered to be approximately: 3-1142 (p125), 1143-1206 (p10) shown in SEQ ID NO: 1 and 2 herein. The putative N-terminus of p80 is amino acid no. 460 (SEQ ID NO: 1 and 2). The start of p30 is also shown in SEQ ID NO. 1 and 2, i.e. amino acid number 1207. The complete sequence of p30 for CSFV Alfort shown in Meyers et al. (Virology 171, 555-567, 1989; Fig. 4 amino acids 2337-2683). The DNA sequences encoding these proteins are also shown in SEQ ID NO. 1 herein and in Meyers et al. (supra).

On infection with CSFV acute, peracute, chronic or clinic invisible symptoms can occur. The severance of the disease is dependent on the infectious load on one hand, on the other hand the age of the animal, the immune competence and total constitution of the animal form important factors.

In the peracute illness, which results in the dead of the animal after three to five days after infection with a highly virulent strain (e.g. the CSFV-Brescia strain), only a fever is observable. In the acute disease state next to a high temperature, leucopenia, conjunctivitis add loss of appetite are observable. In the final stage of the disease central nervous system disturbances occur. Further characteristics are an atrophy of the thymus, cyanosis of the skin and cutaneous haemorrhages, partially caused by a thrombocytopenia. Mortality of the disease in the acute phase is 30-100%.

The chronic disease form is found after infection with mesogenic virus strains. This form is most dangerous when piglets are infected in utero by diaplacental infection. After birth these piglets only survive for 6-8 weeks while they form a source of infection.

Pigs, which survive a postnatal infection, gain lifetime immunity, which probably results from an induction of the humoral immune response. Neutralising antibodies are found after two to three weeks after infection.

Several vaccines have been developed for this economically important disease. Vaccines with inactivated virus give only shortlasting protection and are not used anymore (Biront, P. and Leunen, J., in: Liess, B. (ed.): Classical swine fever and related viral infections. Martinus Nijhoff Publ., Boston pp.

181-200, 1988). Better protection has been established with attenuated viruses obtained by serial passaging in rabbits (C-strains) or in cell cultures (e.g. Thiverval-strain) (Launais, M. et al., Rev. Med. Vet., 123, 1537-1554, 1972; Shimizu Y.,

Jap. J. Trop. Agr. Res. Sci., 13, 167-170, 1980).

However, a disadvantage of these vaccines is that they do not discern between vaccinated animals and animals infected with a field virus. In the European Zommunity, therefore, use of these vaccines is forbidden presently and control of classical swine fever is established by isolation and slaughtering of infected swine.

In recent years some results have been obtained with live vaccines based on recombinant viruses (van Zijl, M. et al., J. Virol. 65, 2761-2765, 1991; Rumenapf. T. et al., J. Virol. 66 ,

589-597, 1991; Hulst, M.M. et al., J. Virol. 67, 5435-5442,

1993). These vaccines have all been based on the expression of structural glycoproteins in recombinant vector viruses. The invention now is directed to a polypeptide comprising a pestivirus T lymphocyte stimulatory protein, or an immunogenically active part thereof.

Such a polypeptide is essentially free from other pestiviral proteins with which it is ordinarily associated.

Specifically the T lymphocyte stimulatory protein is a Classical Swine Fever Virus T lymphocyte stimulatory protein, or an immunogenically active part thereof.

More specifically the polypeptide comprises the CSFV non-structural p10 protein. Because the CSFV proteins are expressed by the virus as a polyprotein which is cleaved subsequent to translation, also polypeptides comprising the p10 protein and its flanking protein(s) p125 and/or p30 or parts thereof are contemplated herein.

More specifically the polypeptide comprises a T cell epitope with the amino acid sequence S-T-A-E-N-A-L-L-V-A-L-F-G-Y-V, most specifically the polypeptide comprises a T cell epitope with the amino acid sequence E-N-A-L-L-V-A-L-F. In general, the term "protein" refers to a molecular chain of amino acids with biological activity. A protein is not of a specific length and can, if required, be modified in vivo or in vitro, by, for example, glycosylation, amidation, carboxylation or phospnorylation; thus, inter alia, peptides, oligopeptides and polypeptiaes are included within the definition.

More particularly, this invention provides polypeptides comprising T-lymphocyte stimulatory proteins, or immunogenically active parts thereof, which comprise the amino acid sequence shown in SEQ ID NO. 2 and their biologically functional equivalents or variants.

lmmunogenically active parts of these polypeptides are those parts of the amino acid sequence that are able to elicit a T cell activation. More specifically this invention includes polypeptides, which comprise the amino acid sequence shown in SEQ ID NO: 6 and still more specifically are polypeptides which comprise the amino acid sequence shown in SEQ ID NO: 4.

The biologically functional equivalents or variants of the proteins specifically disclosed herein are proteins derived from the above noted amino acid sequences, for example by deletions, insertions and/or substitutions of one or more amino acids, but retain one or more immunogenic determinants of CSFV, i.e. said variants nave one or more epitopes capable of eliciting an immune response in a host animal.

It will be understood that, for the particular proteins embraced herein, natural variations can exist between individual virus strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in "The Proteins" Academic Press New York (1979). Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Daynof, M.L., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1976, vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pre, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison

(Science, 227, 1435-1441, 1985) and determining the functional similarity between nomologous proteins. Such. amino acid substitutions of the exemplary emoodiments of this invention are within the scope of the invention as long as the resulting proteins retain their immunoreactivity. The preparation of the proteins according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques.

The organic chemical methods for peptide synthesis are considered to include the coupling of the required amino acids by means of a condensation reaction, either in homogenous phase or with the aid of a so-called solid phase.

The most common methods for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method using activated esters, such as described in The Peptides, Analysis, Synthesis, Biology

Vol. 1-3 (Ed.: Gross, E. and Meienhofer, J.) 1979, 1980, 1981

(Academic Press, Inc.).

Preparation of suitable fragments of above-mentioned peptides according to the invention using the "solid phase" is for instance described in J. Amer. Chem. Soc. 85, 2149 (1963) and Int. J. Peptide Protein Res. 35, 161-214 (1990). The coupling of the amino acids of the peptide to be prepared usually starts from the carboxyl end side. For this method a solid phase is needed on which there are reactive groups or on which such groups can be introduced. This can be, for example, a copolymer of benzene and divinylbenzene with reactive chloromethyl groups, or a polymeric solid phase rendered reactive with hydroxymethyl or amine-function.

A particularly suitable solid phase is, for example, the p- alkoxybezyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl- copolystyrene-18 divinylbenzene resin), described by Wang, J. Am. Chem. Soc. 95, 1326 (1974). After synthesis the peptides can be split from this solid phase under mild conditions. In order to determine if any protein and which was responsible for a T cell mediated effect, CSFV infected target cells were cultured in the following way: from a miniature swine ("NIH-Minipig"; MHC^d/d haplotype) a kidney was removed and cut to pieces under sterile conditions. The organ pieces were rinsed with PBS and pieces were pipetted in a culture flask. They were incubated in culture medium to which a collagenase-dispase solution was added. The cells were rinsed from the tissue with PBS, pelleted and washed and further cultivated.

For obtaining a stable, transformed cell line the cells were transformed with a plasmid having the sequence for the "large T" antigen of SV-40 (Southern, P. and Berg, P., J. Molec. Appl. Genetics., 1 , 327-341, 1982; Fanning, E., J. Virol., 66, 1289-1293, 1992). The MAX-cells thus obtained were selected by culturing with a neomycin analogon G418 (Boehringer) and tested for mycoplasma contamination (Mycoplasma Detection Kit, Boehringer Mannheim). The MAX cells were cultured in DMEM and were infected with CSFV.

To screen the genome of CSFV for epitopes recognized by virus specific T lymphocytes, autologous MAX cells were infected with Vaccinia virus/CSFV recombinants expressing different viral proteins. Successful infection of target cells with the recoitibinants was routinely checked by the cytopathogeinc effect of Vaccinia virus. In addition the expression of CSFV proteins was demonstrated by immunocytocnemical studies and western-blot analysis of infected MAX cells. Effector cells obtained from immunized miniature swine were cocultivated with Vaccinia virus recombinant infecteα radiolabeleo targets (or Vaccinia virus wildtype infected targets as a control) at different effector to target ratios for 4 hours. After centrifugation 100 μl of eacn supernatant were collected and the cnromium release determined as cpm in a gamma radiation counter. Percent specific lysis was calculated by the formula:

Target cell controls were included in the following way: in order to measure spontaneous radioisotope release (spont. cpm), target cells were incubated without effectors. In addition, the total chromium incorporation in target cells was determined (total cpm).

In this way CSFV-specific CTL were identified. For a further characterization of the epitopes responsible for the effects a series of overlapping peptides was synthesized. These peptides were loaded onto the MAX target cells by incubation in wells of round bottom plates for 1 hour. Effector cells were then added to achieve an effector to target cell ratio of 100:1. The plates were centrifuged and incubated and the specific lysis was determined by measuring chromium release as described above.

According to a second aspect of the invention, there is provided a nucleic acid sequence encoding ail or a substantial part, in particular the immunologically active part, o f a purified pestivirus T lymphocyte stimulatory protein, more specifically a CSFV T cell stimulatory protein.

A nucleic acid sequence according to the present invention may be isolated from a particular CSFV strain and multiplier by recombinant DNA techniques including polymerase chain reaction

(PCR) technology or may be chemically synthesized in vitro by techniques known in the art.

The invention further provides isolated and purified nucleic acid sequences encoding the above mentioned proteins of

CSFV. Such nucleic acid sequences are shown in SEQ. ID. NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. It is well known in the art that the degeneracy of the genetic code permits substitution of bases in the codon resulting in another codon but still coding for the same amino acid, e.g. the codon for the amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that, for the expression of a protein with the amino acid sequence shown in SEQ. ID. NO: 2, the nucleic acid sequence may have a codon composition different from the nucleic acid sequence shown in SEQ. ID. NO: 1.

Such a nucleic acid sequence may be operatively linked to various replication sequences with which it is not associated, or linked in nature, resulting in a so-called recombinant vector which can be used for the transfection of a suitable host. Useful recombinant vectors are preferably derived from plasmids, bacteriophages, cosmids or viruses.

Specific vectors or cloning vehicles which can be used to clone nucleic acid sequences according to the invention are known in the art and include inter alia plasmid vectors such as pBR322, the various pUC, pGEM and Bluescript plasmids; bacteriophages, e.g. lambdagt-Wes, Charon 28 and the M13 derived phages or viral vectors such as SV4C, adenovirus or polyoma virus (see also Rodriguez, R.L. and D.7. Denhardt, ed., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988; Lenstra, J.A. et al., Arch. Virol., 110, 1-24, 1990). The methods to be used for the construction of a recombinant vector according to the invention are known to those of ordinary skill in the art and are inter alia set forth in Maniatis, T. et al. (Molecular Cloning A Laboratory Manual, second edition; Cold Spring Harbor Laboratory, 1989).

For example, the insertion of the nuclei-: acid sequence according to the invention into a cloning vector can easily be achieved when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme (s) as complementary DNA termini are thereby produced.

Alternatively, it may be necessary to modify the restriction sites that are produced into blunt ends either by digesting the single-stranded DNA or by filling in the single-stranded termini with an appropriate DNA polymerase. Subsequently, blunt end ligation with an enzyme such as T4 DNA ligase may be carried out.

If desired, any restriction site may be produced by ligating linkers onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site sequences. The restriction enzyme cleaved vector and nucleic acid sequence may also be modified by homopolymeric tailing.

"Transformation", as used herein, refers to the introduction of an heterologous nucleic acid sequence into a host cell, irrespective of the method used, for example direct uptake or transduction. The neterologous nucleic acid sequence may be maintained througn autonomous replication or, alternatively, may be integrated into the host genome. If desired, the recomoinant vectors are prcvided with appropriate control sequences compatible with the designated host. These sequences can regulate the expression of the inserted nucleic acid sequence. In addition to microorganisms, cell cultures derived from multicellular organisms may also be used as hosts. The recombinant vectors according to the invention preferably contain one or more marker activities that may be used to select for desired transformants, such as ampicillin and tetracycline resistance in pBR322, ampicillin resistance and A-peptioe of ß-gaiactosioase in pUC8. A suitable host cell is a microorganism or cell which can be transformed by a nucleic acid sequence encoding a polypeptide or by a recombinant vector comprising such a nucleic acid sequence, and which can, if desired, be used to express said polypeptide encoded by said nucleic acid sequence. The host cell can be of prokaryotic origin, e.g. bacteria such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of eukaryotic origin such as yeasts, e.g. Saccharomyces cerevisiae or higher eukaryotic cells such as insect, plant or mammalian cells, including HeLa cells and Chinese hamster ovary (CHO) cells. Insect cells include the Sf9 cell line of Spodoptera frugiperda (Luckow et al., Biotechnology 6 , 47-55, 1988). Information with respect to the cloning and expression of the nucleic acid sequence of the present invention in eukaryotic cloning systems can be found in Esser, K. et al. (Plasmids of Eukaryotes, Springer-Verlag, 1986).

As host organism also other viruses can be used, which are able to express the inserted pestivirus sequence. Such viruses are commonly denoted vector viruses.

In general, prokaryotes are preferred for the construction of the recombinant vectors useful in the present invention. E. coli K12 strains are particularly useful, especially DH5a or MC1061 strains.

For expression, nucleic acid sequences of the present invention are introduced into an expression vector, i.e. said sequences are operably linked to expression control sequences. Such control sequences may comprise promotors, enhancers, operators, inducers, ribosome binding sites etc. Therefore, the present invention provides a recomoinant vector comprising a nucleic acid sequence encoding a CSFV protein identified above operably linked to expression control sequences, which is capable of expressing the DNA sequences contained therein in (a) transformed host cell(s) or organism. It should be understood, of course, that the nucleotide sequences inserted at the selected site of the cloning vector may include nucleotides which are not part of the actual structural gene for the desired polypeptide, or may include only a fragment of the complete structural gene for the desired protein as long as the transformed host will produce a polypeptide having at least one or more immunogenic determinants of a CSFV protein antigen. When the host cells are bacteria, useful expression control sequences which may be used include the Trp promotor and operator (Goeddel, et al., Nucl . Acids Res., 8 , 4057, 1980); the lac promotor and operator (Chang, et al., Nature, 275, 615, 1978); the outer membrane protein promotor (Nakamura, K. and Inouge, M., EMBO J., 1 , 771-775, 1982); the bacteriophage lambda promotors and operators (Remaut, E. et al., Nucl. Acids Res., 11, 4677-4688, 1983); the A-amylase (B. subtilis) promotor and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell. When the host cell is yeast, illustrative useful expression control sequences include, e.g., A-mating factor. For insect cells the polyhedrin or p10 promotors of baculoviruses can be used (Smith, G.E. et al., Mol. Cell. Biol. 3 , 2156-65, 1983 J. When the nost cell is of mammalian origin illustrative useful expression control sequences include the SV-40 promotor (Berman, P.W. et al., Science, 222, 524-527, 1983) or the metallothionein promotor (Brinster, R.L., Nature, 296, 39-42, 1982) or a heat snock promotor (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949- 53, 1985). Alternatively, expression control sequences present in CSFV may also be applied. For maximizing gene expression, see also Roberts and Lauer (Methods in Enzymology, 65, 473, 1979). Therefore, the invention also comprises (a) host cell(s) or organism(s) having a nucleic acid sequence or a recombinant nucleic acid molecule or a recombinant vector described above, capable of producing the pestivirus protein by expression of the nucleic acid sequence. Immunization of animals against pestivirus infection, especially swine against CSFV, can be achieved by administering to the animals a polypeptide according to the invention in an immunologically relevant context as a so-called subunit vaccine. The subunit vaccine according to the invention may comprise a polypeptide in a pure form, optionally in the presence of a pharmaceutically acceptable carrier. The polypeptide can optionally be covalently bonded to a non-related protein, which can be of advantage in the purification of the fusion product. Examples are ß-galactosidase, protein A, prochymosine, blood clotting factor Xa, etc.

In some cases the ability to raise protective immunity using these polypeptides per se may be low. Small fragments are preferably conjugated to carrier molecules in order to raise their immunogenicity. Suitable carriers for this purpose are macromolecules, such as natural polymers (proteins like key hole limpet hemocyanin, albumin, toxins), synthetic polymers like polyamino acids (polyiysine, polyalanine), or micelles of amphiphilic compounds like saponins and paimitinic acid. Alternatively these fragments may be provided as polymers thereof, preferably linear polymers.

If required, the proteins according to the invention which are to be used in a vaccine can be modified in vitro or in vivo, for example by glycosylation, amidation, carboxylation or phosphorylation.

The immunological system will even be more effectively- triggered when the vaccine comprises the polypeptides as presented in an MHC molecule by an antigen presenting cell (APC). Antigen presentation can be achieved by using monocytes, macrophages, interdigitating cells, Langerhans cells and especially dendritic cells, loaded with one of the peptides of the invention. Loading of the APC's can be accomplished by bringing the polypeptides of the invention into or in the neighbourhood of the APC, but it is more preferable to let the APC process the complete antigen. In this way a presentation is achieved which mimicks the in vi vo situation the most realistic. Furthermore the MHC used by the cell is of the type which is suited to present the epitope.

An overall advantage of using APC's for the presentation of the epitopes is the choice of APC cell that is used in this respect. It is known from different types of APC's that there are stimulating APC's and inhibiting APC's.

Prefered are the listed cell types, which are so-called 'professional' antigen presenting cells, characterized in that they have co-stimulating molecules, which have an important function in the process of antigen presentation. Such co-stimulating molecules are, for example, B7, CTLA-4, CD70 or heat stable antigen.

Fibroblasts, which have also been shown to be able to act as an antigen presenting cell, lack these co-stimulating molecules .

It is also possible to use cells already transfected with a cloning vehicle harbouring the information for the polypeptides of the invention and which are cotransfected sith a cloning vehicle which comprises the nucleotide sequence for an MHC molecule. These cells will act as an antigen presenting cell and will present pestivirus epitopes in the MHC molecules which are expressed on their surface. It is envisaged that this presentation will be enhanced, when the cell is also capable of expressing one of the above-mentioned co-stimulating molecules, or a molecule with a similar function. This expression can be the result of transformation or transfection of the cell with a third cloning vehicle having the sequence information coding for such a co-stimulating molecule, but it can also be that the cell already was capable of production of co-stimulating molecules. In stead of a vaccine with these cells, which next to the desired expression products, also harbour many elements which are also expressed and which can negatively affect the desired immunogenic reaction of the cell, it is also possible that a vaccine is composed with liposomes which expose MHC molecules loaded with peptides, and which, for instance, are filled with lymphokines. Such liposomes will trigger a immunologic T cell reaction. By presenting the peptide in the same way as it is also presented in vi vo an enhanced T cell response will be evoked. Furthermore, by the natural adjuvant working of the, relatively large, antigen presenting cells also a B cell response is triggered. This B cell response will a.o. lead to the formation of antibodies directed to the peptide-MHC complex. It is this naturally occurring phenomenon which is enlarged by the vaccination of APC's already presenting the peptides of the invention. By enlarging not only an enlarged T cell response will be evoked, but also a B cell response which leads to antibodies directed to the MHC-peptide complex will be initiated.

An alternative to subunit vaccines is live vaccines. A nucleic acid sequence according to the invention is introduced by recombinant DNA techniques into a nost cell or organism

(e.g. a bacterium or virus) in such a way that the recombinant host is still able to replicate, thereby expressing a polypeptide coded by the inserted nucleic acid sequence and eliciting an immune response in the infected host swine.

A preferred emoodiment of the present invention is a recombinant vector virus comprising a neterologous nucleic acid sequence described above, capable of expressing the DNA sequence in (a) host cell(s or host swine infected with the recombinant vector virus. The term "heterologous" indicates that the nucleic acid sequence according to the invention is not normally present in nature in the vector virus.

Furthermore, the invention also comprises (a) host cell(s) or cell culture infected with the recombinant vector virus, capable of producing the CSFV protein by expression of the nucleic acid sequence.

For example the well known technique of in vivo homologous recombination can be used to introduce an heterologous nucleic acid sequence according to the invention into the genome of the vector virus.

First, a DNA fragment corresponding with an insertion region of the vector genome, i.e. a region which can be used for the incorporation of an heterologous sequence without disrupting essential functions of the vector such as those necessary for infection or replication, is inserted into a cloning vector according to standard recDNA techniques. Insertion-regions have been reported for a large number of microorganisms (e.g. EP 80,806, EP 110,385, EP 83,286, EP 314,569, WO 88/02022, WO 88/07086, US 4,769,330 and US 4,722,848). Second, if desired, a deletion can be introduced into the insertion region present in the recombinant vector molecule obtained from the first step. This can be achieved for example by appropriate exonuclease III digestion or restriction enzyme treatment of the recombinant vector molecule from the first step.

Third, the heterologous nucleic acid sequence is inserted into the insertion-region present in the recombinant vector of the first step or in place of the DNA deleted from said recombinant vector. The insertion region DNA sequence should be of appropriate length as to allow homologous recombination with the vector genome to occur. Thereafter, suitable cells can be infected with wild-type vector virus or transformed with vector genomic DNA in the presence of the recombinant vector containing the insertion flanked by appropriate vector DNA sequences whereby recombination occurs between the corresponding regions in the recombinant vector and the vector genome. Recombinant vector progeny can now be produced in cell culture and can be selected for example genotypically or phenotypically, e.g. by hybridization, detecting enzyme activity encoded by a gene co-integrated along with the heterologous nucleic acid sequence, or detecting the antigenic heterologous polypeptide expressed by the recombinant vector immunologically.

Next, these recombinant microorganisms can be administered to swine for immunization whereafter they maintains themselves for some time, or even replicate in the body of the inoculated animal, expressing in vivo a polypeptide coded for by the inserted nucleic acid sequence according to the invention resulting in the stimulation of the immune system of the inoculated animal. Suitable vectors for the incorporation of a nucleic acid sequence according to the invention can be derived from viruses such as pox viruses, e.g. vaccinia virus (EP 110,385, EP 83,286, US 4,769,330 and US 4,722 848,, herpes viruses such as Aujeszκy virus (van Zijl, M . et al., J. Virol. 62(6), 2191-2195, 1988/, porcine respiratory syncitiae virus, adeno virus or influenza virus, or bacteria sucn as E. coll, Streptococcus suis, Actinooacillus pleuropneumoniae o r speci f i c Salmonella species . With recombinant microorganisms of this type, the polypeptide synthesized in the host animal can be exposed as a surface antigen. In this context fusion of the polypeptide with OMP proteins, or pilus proteins of for example E. coli or synthetic provision of signal and anchor sequences which are recognized by the organism are conceivable. It is also possible that the pestivirus poiypeptiαe, if desired as part of a larger whole, is released insiαe the animal to be immunized. In all of these cases it is also possible that one or more immunogenic products will find expression which generate protection against various pathogens and/or against various antigens of a given pathogen. A vector vaccine according to the invention can be prepared by culturing a recombinant bacterium or a host cell infected with a recombinant vector comprising a nucleic acid sequence according to the invention, whereafter recombinant bacteria or vector containing cells and/or recombinant vector viruses grown in the cells can be collected, optionally in a pure form, and formed into a vaccine optionally in a lyophilised form.

Host cells transformed with a recombinant vector according to the invention can also be cultured under conditions which are favourable for the expression of a polypeptide coded by said nucleic acid sequence. Vaccines may be prepared using samples of the crude culture, host cell lysates or host cell extracts, although in another embodiment more purified polypeptides according to the invention are formed into a vaccine, depending on its intended use. In order to purify the polypeptides oroouced, host cells transformed with a recombinant vector according to the invention are cultured in an adequate volume and the polypeptides produced are isolated from such ceils, or from the medium if the protein is excreted. Polypeptides excreted into the medium can be isolated and purified by standard techniques, e.g. salt fractionation, centrifugation, ultraflitration, chromatograpny, gel filtration or immuno affinity chromatography, whereas intracellular polypeptides can be isolated by first collecting said cells, disrupting the cells, for example by sonication or by other mechanically disruptive means such as French press, followed by separation of the polypeptides from the other intracellular components and forming the polypeptides into a vaccine. Cell disruption could aiso ce acnieved by cnemical e.g. EDTA or detergents such as Triton X114 cr enzymatic means, sucn as lvsozvme digestion. It is also possible to vaccinate animals with the "nude"

DNA, i.e. the nucleic acids as defined above without any regulatory sequences. This DNA then will be incorporated in the genome of the vaccinated animal and thus express the polypeptide of the invention.

The vaccines according to the invention can be enriched by numerous compounds which have an enhancing effect on the initiation and the maintenance of both the T cell and the B cell response after vaccination.

In this way addition of cytokines to the vaccine will enhance the T cell response. Suitable cytokines are for instance interleukines, such as IL-2, IL-4, IL-7, or IL-12, GM-CSF, RANTES, tumor necrosis factor and interferons, such as

IFN-gamma.

Antibodies or antiserum directed against a polypeptide according to the invention have a potential use in passive immunotherapy, diagnostic immunoassays and generation of anti-idiotypic antibodies.

The vaccine according to the invention can be administered in a conventional active immunization scheme: single or repeated administration in a manner compatible with the dosage formulation, and in such amount as will be prophylactically effective, i.e. the amount of immunizing antigen or recombinant microorganism capable of expressing said antigen that will induce immunity in swine against challenge by virulent CSFV. It can also be given in addition to a conventional (B lymphocyte directed) vaccination to enhance the immunity caused by such a vaccination. Immunity is defined as the induction of a significant level of protection in a population of animals after vaccination compared to an unvaccinated group.

For live viral vector vaccines the dose rate per animal may range from 10²-10⁸ pfu. A typical suounit vaccine according to the invention comprises 1 μg - 1 mg of the protein according to the invention . Such vaccines can be administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally or intranasally .

Additionally the vaccine may also contain an aqueous medium or a water containing suspension, often mixed with other constituents in order to increase the activity and/or the shelf life. These constituents may be salts, pH buffers, stabilizers (such as skimmed milk or casein hydrolysate), emulsifiers, adjuvants to improve the immune response (e.g. oils, muramyl dipeptide, aluminium. hydroxide, saponin, polyanions and amphipatic substances) and preservatives. It is clear that a vaccine according to the invention may also contain immunogens related to other pathogens of swine, or may contain nucleic acid sequences encoding these immunogens, like antigens of Actinobacillus pleuropneumoniae, Pseudorabies virus, Porcine Influenza virus, Porcine Parvovirus, Streptococcus suis, Transmissible Gastroenteritisvirus, Rotavirus, Escherichia coll, Erysipelothriy rhusiopathiae, Pasteurella multocida and Bordetella bronchiseptica.

The invention also relates to an "immunocnemical reagent", which reagent comprises a protein according to the invention. The term "immunochemical reagent" signifies that the protein according to the invention is bound to a suitaoie support or is provided with a labelling substance. The supports that may be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a aye sol, a metal sol or metal compound as sol particle. Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle. A nucleic acid sequence according to the invention can also be used to design specific probes for hybridization experiments for the detection of pestivirus related nucleic acids in any kind of tissue. The present invention also comprises a test kit comprising said nucleic acid sequence useful for the diagnosis of pestivirus, specifically CSFV infection.

The invention also relates to a test kit to be used in an immunoassay, this test kit containing at least one immunochemical reagent according to the invention. The immunochemical reaction which takes place using this test kit is preferably a sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.

For carrying out a sandwich reaction, the test kit can consist, for example, of a polypeptiαe according to the invention bonded to a solid support, for example the inner wall of a microtest well, and either a labelled polypectide according to the invention or a labelled anti-antioody.

The invention is illustrated by the following examples.

EXAMPLES

EXAMPLE 1: Identification of CSFV specific cytotoxic

lymphocytes.

Peripheral blood leukocytes were isolated from inbred miniature swine of MHC^d,/d haplotype after immunization with CSFV-strain Riems (4-10⁵ TCID₅₀ i.m.) following repeated challenges with 2-10⁷ TCID₅₀ CSFV-strain Alfort and a final challenge with 3 ml intranasally of a serum (1·10⁵ TCID₅₀) obtained from a CSFV-strain Brescia infected pig. The cells were seeded in 96-well round bottom microtiter plates at a concentration of 1-2·10⁵ 6ells per microculture in RPMI medium (10% FCS) and simultaneously restimulated with infectious virus (5·10⁵ TCID₅₀/ml CSFV-Alfort) for 3 to 5 days.

Culturing of infected target cells was done in the following way: from a miniature swine ("N1H-Minipig"; MHC^d/d haplotype) a kidney was removed and cut to pieces under sterile conditions. The organ pieces were rinsed with PBS and 30-100 pieces were pipetted in a culture flask (25 cm²). They were incubated in culture medium (10 FCS) to which a collagenase- dispase solution (stocksolution of 2.5 mg/ml diluted 1:6 in medium) was added. The cells were rinsed from the tissue with PBS, pelleted (6 min, 750 g) and washed twice in PBS. They were cultivated in DMEM (10% FCS) in culture flasks (25 and 75 cm²).

For obtaining a stable, transformed cell line of target cells the kidney cells were transformed with a plasmid having the sequence for the "large T" antigen of SV-40 (Southern, P. and Berg, P., J. Molec. Appi . Genetics., 1, 327-341, 1982;

Fanning, E., J. Virol., 66, 1289-1292, 1992:. For this purified

SV-40 plasmid pSV-3 nee (from. Dr T.C. Mettenleiter, BFAV

Tubingen) was mixed with iipofectin to a DNA-lipofectin ratic of 1:4 and incubated for 15 min at room temperature. Of a largely (80%) confluent kidney cell culture the culture medium was removeα and 2 ml OptiMEM-Medium (Gibco) was added. The lipofectin/DNA suspension was then added dropwise and the culture was initially incubated for 12 hours at 37°C. Then the medium was replaced by cloning medium (DMEM, F10 (Gibco) and F12(Gibco) in a 2:1:1 ratio) with 10% FCS (foetal calf serum) and cultivated at 37°C. The MAX-cells thus obtained were selected by culturing with a neomycin analogon G418 (Boehringer) and tested for mycoplasma contamination (Mycoplasma Detection Kit, Boehringer Mannheim).

1·10⁶ target cells were infected with CSFV-Alfort at a m.o.i. of 0.5. 48 hours after infection the cells were trypsinated and collected in a 200 μl CTL assay medium (RPMI 1640 (Gibco), 31 FCS). The target cells were labelled with 100 μCi of Na₂ ⁵¹CrO₄ for 90 min., washed three times and resuspended in CTL assay medium at a final concentration of 1·10⁴ cells/ml.

Effector cells were diluted to achieve different effector:target ratios and were added to triplicate wells in 100 μl volumes. 1·10³/well target cells were added to the effector cells. The plates were centrifuged at 100g for 5 minutes and incubated for 4 hours at 37°C. After centrifugation at 600g for 10 minutes, 100 μl of each supernatant were collected and the chromium release determined as cpm in a gamma radiation counter. Percent specific lysis was calculated by the formula:

Target cell controls were included in the following way: in order to measure spontaneous radioisotope release t spont. cpm) , target cells were incubated without effectors. In addition, the total chromium incorporation in target cells was determined (total cpm).

Results

Lysis of CSFV-infected target cells by cytotoxic T lymphocytes was higher than in the control group (figure 1). A more than 30% higher specific lysis was reached with a relatively low effector to target cell ratio (12:1), indicating a high rate of CSFV specific cytotoxic T lymphocytes.

EXAMPLE 2 : Cytotoxic T lymphocytes mediated lysis of target cells expressing truncated proteins. 1-10⁶ MAX cells (obtained as described in Example 1) were infected with different Vaccinia virus/CSFV recombinants at a multiplicity of infection of 2.0 for 16 hours. Figure 2 shows the relative positions of the proteins of CSFV and the identification of the Vaccinia virus/CSFV recombinants obtained.

Cells which showed a moderate cytopathic effect, were trypsinated and collected in 200 μl CTL assay medium. The target cells were labelled with 100 μCi of Na2⁵¹CrO₄ for 90 min, washed three times and resuspended in CTL assay medium at a concentration of 1·10⁴ cells/ml prior to use in the assay.

The assay was conducted in the same way as described in Example 1.

Results

The results of the chromium release assays indicated that CSFV-specific CTL recognize only target cells infected with the Vaccinia virus/p125S recombinant. Surprisingly, neither target cells expressing viral structural proteins nor cells expressing the autoprotease were lysed by the specific cytotoxic T lympnocytes (figure 3). From the peptide products produced by the Vaccinia virus/p125S recombinant it seemes that the 80 kD subunit or the p10 protein are responsible for the recognition (figure 4, upper middle graph). Narrowing down this area it appears that Vaccinia virus/CSFV recombinant p80/VZ still shows specific lysis, while p80/VA and p80/VX are ineffective. This means (see figure 2; the nucleotide positions refer to the sequence of CSFV Alfort shown by Meyers et al., Virology 171, 555-567, 1989) that the T cell specific epitope is situated near the cleavage site between p125 and p10. The region identified as the region in which the epitope is harboured (the region still present in the recombinant p80/VZ and not present in p80/VA and p80/VX) is situated from amino acid position 2223 to 2285

(according to the sequence of CSFV Alfort (Meyers et al. supra), i.e. amino acid positions 1093 to 1155 of SEQ ID NO:2). Data obtained by N-terminal protein sequencing of p10 of BVDV-strain cp7 reveal that the cleavage site between p125 and p10 of CSFV-Alfort is located between amino acid positions 2272 and 2273 of CSFV-Alfort, i.e. between nucleotide positions 3426 and 3427 of SEQ ID NO : 1.

EXAMPLE 3 : Identification of a T cell epitope recognized by

CSFV-specific CTL.

To determine the epitope recognized by virus specific CTL, nonapeptides and pentadecapeptides were synthesized overlapping by 8 and 12 amino acids, respectively, covering the amino acid region from positions 1093 to 1155 of SEQ ID NO:2.

1-10⁶ target cells were labelled with 100 μCi of Na2⁵¹CrO₄ for 90 min, washed three times and resuspended in CTL assay medium at a final concentration of 2.10⁴ cells/ml. 50 μl of target cell suspension was added to triplicate wells of 96 well round bottom plates. The target cells were loaded with peptide by incubation with 100 μl peptide solution (5 mg/ml stock solution in DMSO; 1:4000 to 1:500C dilutee in RPMI medium, resulting in approximately 50 to 125 ng/10³ target cells) for 1 hour. 1-10⁵ effector cells were added to each well in 50 μl volumes to achieve an effector to target cell ratio of 100:1.

Chromium release assay was done in a way similar to Example 1 and 2.

Results

Specific lysis of target cells loaded with different peptides is shown in figures 5 and 6. In figure 5 it is shown that the nonapeptide E-N-A-L-L-V-A-L-F causes the largest percentage of specific lysis, while in figure 6 the pentadecapapetide S-T-A-E-N-A-L-L-V-A-L-F-G-Y-V resulted in a significantly higher lysis of target cells compared to the target cells incubated with other pentadecapeptides. This pentadecapapetide contains the sequence of the nonapeptide identified already in figure 5. Nevertheless, CTL lysed target cells loaded with the nonapeptide more efficiently than target cells loaded with the pentadecamer, suggesting that nonapeptides have a higher affinity to porcine MHC-I molecules than pentadecapeptides.

EXAMPLE 4: Crossreactivity of CSFV-specific CTL. Experiments regarding the crossreactivity of CSFV-specific CTL were performed using target cells infected with different CSFV-strains at a m.o.i. of 0.5 for 48 hours (performed in accordance to Example 1) or infected with the Vaccinia virus recombinants at a m.o.i. of 2.0 for 16 hours (performec in accordance to Example 2). The Vaccinia virus recombinants contained the coding sequences for the non-structural proteins p125, p10 and the N-terminal part of p30 of CSFV-Aifort Tubingen (Vac-p125S) or the corresponding proteins of BVDV strain cp7 (Vac-p120S). The CTL activity of CSFV-sensitizeo effector cells was determined in chromium release assays using the infected MAX cells as targets. For control non-infected and vaccinia virus wildtype (Vac-WR) infected targets were used.

Results

The results of both the experiments with CSFV-strains and with vaccinia virus recombinants are depicted in Table I. From these results it can be seen that CSFV specific CTL were capable of lysing target cells infected with different CSFV-strains. It is also shown that CSFV specific CTL were capable of lysing target cells expressing nonstructural proteins of a BVDV-strain, thereby suggesting that these nonstructural proteins harbour a T cell specific epitope which may be conserved for pestiviruses.

LEGENDS TO THE FIGURES

Fig, 1:

Results of chromium release assay indicating CSFV specific lysis of target cells by cytotoxic T cells. Open circles indicate non infected control target cells, full circles indicate CSFV infected target cells. The 1 specific lysis is indicated on the Y-axis, while the X-axis shows the ratio of effector to target cells.

Fig. 2:

Vaccinia virus/CSFV recombinants used for infection of autologous target cells. The relative positions of the N-terminai autoprotease (N^pro), the structural proteins core (C), E0, E1, E2 and the nonstructural proteins p125, p30 and p133 on the CSFV polyprotein as well as their processing products are indicated. In addition, the viral proteins and truncated p125-proteins expressed as Vaccina virus/CSFV recombinants are shown. The amino acid positions indicated are in accordance to the sequence of CSFV-Alfort.

Fig. 3:

CTL mediated lysis of target cells infected with different Vaccinia virus/CSFV recomoinants. MAX-ceils were infected with Vaccinia virus wildtype (Vacc-WR, open circles) and the Vaccinia virus/CSFV recombinants Vacc-N^proC (open squares), Vacc-E012 (closed squares) or Vacc-p125S (closed circles) and used as target cells in chromium release assays against CSFV- specific CTL. Axes identical to figure 1.

Fig. 4:

CTL mediated lysis of target cells expressing truncated nonstructural proteins. Presentation of data analog to figure

3. Fig . 5 :

Lysis of peptide target cells by CSFV-specific CTL. Radiolabelled MAX-cells were incubated with different nonapeptides. The bars indicate percent specific lysis by an effector to target ratio of 100 to 1. Controls: specific lysis of CSFV-infected and non-infected target cells.

Fig. 6:

Lysis of pentadecapeptide loaded target cells. Legends as in figure 5.

Claims

1. A vaccine for the protection of swine against classical swine fever (CSF), characterized in that it comprises CSF virus polypeptide p10 or an immunogenically active part thereof, together with a pharmaceutically acceptable carrier .

2. A vaccine according claim 1, characterized in that the p10 polypeptide has an amino acid sequence shown in SEQ ID NO: 2.

3. A vaccine for the protection of swine against CSF, characterized in that it comprises a recombinant micro- organism harbouring a DNA sequence encoding the polypeptide defined in claim 1 or 2, together with a pharmaceutically acceptable carrier.

4. A vaccine according to claim 3, characterized in that the recombinant micro-organism is a recombinant vector virus, preferably recombinant pseudorabies virus.